p53, also known as tumor protein 53, is a tumor suppressor gene involved in the prevention of cancer, often referred to as the gatekeeper or guardian of the genome (Levine, Cell 1997, 88:323-331). The p53 gene encodes for a transcription factor that is normally quiescent, and becoming activated when the cell is stressed or damaged, such as when DNA damage incurred from a mutagen. If the cell is stressed or damaged, p53 acts to limit the damage, or barring that, trigger the apoptotic pathway so the damaged cell is eliminated and no longer a threat to the organism (Vogelstein et al., Nature 2000, 408:307-310). An analysis of different cancers showed that p53 is mutated in about 50% of human cancers (Hollstein et al., Nucleic Acids Res. 1994, 22:3551-3555: Hollstein et al., Science 1991, 253(5015): 49-53). Humans who are heterozygous for p53, with only a single functional copy, will develop tumors early in adulthood, a disorder known as Li-Fraumeni syndrome (Varley et al., Hum. Mutat. 2003, 21(3):313-320). However, as much as p53 regulates the cell's fate, p53 is regulated by another protein known as MDM2.
Double minute 2 protein (MDM2) was discovered as a negative regulator of p53 (Fakharzadeh et al., EMBO J. 1991, 10(6):1565-1565). MDM2 encodes an E3 ligase containing a p53 binding domain and a nuclear export signal sequence, and upon complexing with p53, removes it from the nucleus and ubiquitinylates it, which promotes the degradation of the p53 protein via the ubiquitin-proteosome pathway (Haupt et al., Nature 1997, 387(6630):296-299; Piette et al., Oncogene 1997 15(9):1001-1010). In addition, MDM2 directly inhibits the activity of p53 by binding to the p53 transactivation domain, also preventing p53 mediated gene expression (Wu et a., Genes Dev. 1993, 7:1126-1132). Thus, MDM2 regulates p53 in multiple ways.
MDM2 is overexpressed in a number of cancers, for example, liposarcoma, glioblastoma, and leukemia (Momand et al., Nucleic Acids Res. 1998, 26(15):3453-3459). Overexpression of MDM2 can interfere with the activities of p53, preventing apoptosis and growth arrest of the tumor (de Rozieres et al., Oncogene 2000, 19(13):1691-1697). Overexpression of MDM2 correlates with poor prognosis in glioma, and acute lymphocytic leukemia (Onel et al., Mol. Cancer Res. 2004, 2(1):1-8).
As MDM2 is an inhibitor of p53, therapeutics which prevent the binding of MDM2 to p53 would prevent the degradation of p53, allowing free p53 to bind and mediate gene expression in cancer cells, resulting in cell cycle arrest and apoptosis. There are previous reports of small molecule inhibitors of the p53-MDM2 interaction (Vassilev et al., Science, 2004, 303(5659):844-888; Zhang et al., Anticancer drugs, 2009 20(6):416-424; Vu et al., Curr. Topics Microbiol. Immuno., 2011, 348:151-172). The mode of binding of these compounds and a crystal structure of the human MDM2-Nutlin complex as well as a scaffold and pockets of the p53 binding site on MDM2 are also known (Vassilev, supra). The first of these MDM2 inhibitors, known as the Nutlins, bind MDM2 and occupy the p53 binding pocket, preventing the formation of the MDM2-p53 complex. This leads to less degradation of the p53 protein, and expression of p53 target genes. Cancer cell lines treated with Nutlins showed growth arrest and increased apoptosis. For example, the SJSA-1 osteosarcoma line contains amplified copies of the MDM2 gene. Treatment of this line with Nutlin-3 reduced proliferation and increased apoptosis (Vassilev et al., Science, 2004, 303(5659):844-888). The SJSA-1 cell line was used in creating xenographs in mouse. Administration of Nutlin-3 reduced xenograft growth by 90%. To investigate the effect the Nutlin compounds had on non-cancerous cells, human and mouse normal fibroblasts were treated with Nutlin-3 and while the proliferation of the cells was slowed, they retained their viability (Vassilev, supra).
Finding biomarkers which indicate which patient should receive a therapeutic is useful, especially with regard to cancer. This allows for more timely and aggressive treatment as opposed to a trial and error approach. In addition, the discovery of biomarkers which indicate that cells continue to be sensitive to the therapy after administration is also useful. These biomarkers can be used to monitor the response of those patients receiving the therapeutic. If biomarkers indicate that the patient has become insensitive to the treatment, then the dosage administered can be increased, decreased, completely discontinued or an additional therapeutic administered. As such, there is a need to develop biomarkers associated with MDM2 inhibitors. This approach ensures that the correct patients receive the appropriate treatment and during the course of the treatment the patient can be monitored for continued MDM2 inhibitor sensitivity.
In the development of MDM2 inhibitors, specific biomarkers will aid in understanding the mechanism of action upon administration. The mechanism of action may involve a complex cascade of regulatory mechanisms in the cell cycle and differential gene expression. This analysis is done at the pre-clinical stage of drug development in order to determine the particular sensitivity of cancer cells to the MDM2 inhibitor candidate and the activity of the candidate. Of particular interest in the pharmacodynamic investigation is the identification of specific markers of sensitivity and activity, such as the ones disclosed herein.